(1) Field of the Invention
This invention relates to the techniques to recover macromolecules, specifically to recover DNA molecules from agarose gels.
(2) Description of the Prior Art
In laboratories, DNAs are frequently cut into fragments with enzymes and separated by agarose gel electrophoresis. In the electrophoresis process, charged DNA fragments migrate towards the positive electrode through agarose gel matrix submerged in electrophoresis buffer. A smaller fragment, having less resistance in an agarose gel, migrates faster than a larger one and thus is separated from the latter. DNA recovery from agarose gels is an essential step in the recombinant DNA technology. In the 1970s, protocols for DNA recovery from agarose gels were published. Among the protocols, the following were relatively successful: the freeze-squeeze method (Thuring, R. W. J., et al., Anal. Blochem. 66, 213-220, 1975), gel dissolution using potassium iodide (Blin, N., et al., FEBS lett. 53, 84-86, 1975), electroelution (Wienand, U., et al. FEBS Lett. 98, 319-323, 1978), and the techniques involving binding of DNA to powdered glass. The freeze-squeeze method, less efficient especially for larger fragments, is seldom used nowadays. The other methods have been modified and improved. The most used methods for DNA recovery from agarose gels are described in detail in "CURRENT PROTOCOLS IN MOLECULAR BIOLOGY" (Ausubel, F. M., et al., John Wiley & Sons, New York, 1991, pp. 2.6.1-2.7.5) and "MOLECULAR CLONING" (Samgrook, J., et al. Cold Spring Harbor Laboratory Press, 1989, pp. 6.22-6.46), two standard manuals used in almost all labs involving in recombinant DNA techniques or molecular biology. Six commonly used methods are listed below (three methods described in the two lab manuals and three commercially available products):
Electroelution into dialysis bags, PA1 Electrophoresis onto diethylaminoethyl (DEAE)-cellulose membrane, PA1 Low-melting temperature agarose gel method, PA1 Gelase digestion method (Epicentre Technologies, Madison, Wisc.) PA1 GENECLEAN kit (Bio 101 Inc. La Jolla, Calif.), PA1 SpinBind method (FMC BioProducts, Rockland, Me.).
Electrophoresis onto DEAE-cellulose method is not suitable for very large or very small DNA fragments. Low-melting gel method is less reproducible and labor intensive. Gelase digestion method requires a long incubation time (12-16 hours). The procedures of GENECLEAN kit and SpinBind are complicated and very technique dependent. In addition, these methods require temperature-controlled water bath, or chromatographic columns, or additional chemicals that are toxic to the environment.
Electroelution into dialysis bag method is particularly appropriate for the recovery of a wide size range of DNA fragments and large amount of DNA, and it is very reproducible and reliable. But the method is very inconvenient, and a significant amount of DNA can be lost. Therefore it is recommended to be used only when necessary for large DNA fragment recovery which is inefficient by other techniques (Current Protocols, and Molecular Cloning). The inconvenience and the significant DNA loss of this method are due to the awkward procedure and the large volume of buffer in which DNAs are eluted.
An article in the journal of Analytical Biochemistry 124, 299-302 (1982), U.S. Pat. No. 4,725,348 to Diekmann (1988), U.S. Pat. No. 4,948,481 to Mullner (1990), U.S. Pat. No. 4,608,147 to Clad (1986), and U.S. Pat. No. 4,964,961 to Brautigam et al. (1990) disclosed several apparatuses for electroeluting charged macromolecules. They are all rather complicated and not very easy to use. Therefore they are seldom seen in laboratories.
All the above methods are not satisfactory. Simpler and better methods are long awaited. The current invention meets this demand. In this invention, an elution device, a disposable cassette, is designed based on the same principle of electroelution into dialysis bag method. Since the procedure is simplified and elution volume is minimized, the new invention retains all advantages and eliminates the disadvantages of the method of electroelution into dialysis bag.